Summary of Work: We often use the powerful bacteriophage T4 model system to study mechanisms of mutagenesis and DNA repair. We have developed a new T4 mutation reporter gene, the rI gene, which is useful for mutation studies because it is not involved in DNA metabolism, is small enough to be accommodated by a single DNA sequencing lane, and is sensitive to missense mutations. The rate of spontaneous mutation is normal in this gene. A striking mutational hot-spot is observed, consisting of three adjacent base pair substitutions that arise simultaneously. This mutation is associated with an imperfect palindrome of considerable length, and the mutation occurs at the highest rate ever observed for a palindrome-driven complex mutation. We also observed a single rare mutation consisting of six base pair substitutions scattered over 17 base pairs; in this case, the associated imperfect palindrome strongly overlaps itself, providing information useful for modeling the formation of complex mutations.